HELP making 6X SDS loading buffer for SDS PAGE

[pombe13]

Hello,

 

I need some tips on how to make a 6X SDS loading buffer for use in SDS PAGE.

My biggest problem is getting the SDS into solution.

Last time I made it, I heated the solution on the lowest possible setting on our stir/heating plate to get it into solution. Once the SDS appeared to be dissolved, I turned the heat off and added the DTT and bromophenol blue. While I was aliquotting the solution for storage, it almost seemed to be becoming more solid. Indeed, I just removed an aliquot from our -20C and it is impossible to pipette and seems to be more of a gel-like solid than a liquid. (Our 6X loading buffer has always been thick in the past, but not this thick). I also have tried making the buffer without heating at all, and the solution becomes super thick even quicker.

 

This is the recipe we use for 10mL:

 

3.75mL 1M Tris pH 6.8

1.2g SDS (solid)

6mL glycerol (100% stock)

0.006g bromophenol blue

0.462g DTT

 

Any help would be greatly appreciated, or if anyone has a protocol for making a 6X SDS loading buffer that works for them I would also appreciate that! (Please tell me how you get the SDS to dissolve without turning the solution into a solid!)

[CharonY]

The high viscosity of the solution is not due to precipitation of SDS but due to the glycerol. Even if SDS precipitates, the solution would still be liquid (with SDS flakes floating around).

 

Generally I would also avoid heating and at that concentration also unnecessary. Did you dissolve SDS only in buffer (i.e. without glycerol)?

Alternatively you could reduce the SDS concentration a bit (common values are somewhere between 6-10%) as well as glycerol (down to 40%). Third possibility is to create less strongly concentrated loading dye, but if working with dilute protein samples that may not be ideal (though 5x concentrated is still very close and you are not as close to the solubility limit of SDS).

 

Also, if you freeze your buffer, do not forget to let it get to RT before pipetting. For the most part I do not recall specific issues with creating 6x loading buffers, but for various reasons I started using 5x for most standard applications.